The advent of recombinant DNA technology has made it possible to insert genetic information from one species into another species in such a manner that expression of the "foreign" genetic information can be effected under controlled conditions in the host organism. Thus, it has become possible to insert genes coding for a variety of useful polypeptides and proteins into common microorganisms, grow up the "transformed" microorganisms in industrial scale fermentors and produce large quantities of the polypeptides or proteins by controlled expression of the inserted genetic information. In this manner, for example, a variety of proteins having medically useful properties, such as interferons, insulin, interleukin-2, human tissue type plasminogen activator or the like, and industrially useful enzymes, such as rennin, have been produced.
Until now, most of the commercial applications of recombinant DNA technology have involved the use of Escherichia coli as a host microorganism. This common enteric bacteria has been thoroughly studied and is well suited for the genetic manipulations necessary to the insertion of foreign genetic information. Unfortunately, E. coli has a number of serious drawbacks associated with its use as a host microorganism for commercial production. The organism produces endotoxins which represent a serious threat of contamination to the desired product. The threat of contamination by endotoxins is a particularly serious problem in the production of products intended for human or animal consumption, such as medicines and hormones. Scrupulous care must be taken to assure that all traces of endotoxin have been removed from the product prior to use.
E. coli is not well suited for the secretion of foreign gene products from the cell. The expressed product is generally sequestered within the cell, necessitating cell lysis for recovery of the desired product. Recovering the product in this manner often requires laborious and expensive purification procedures since the desired product must be separated from numerous other intracellular proteins produced by the organism. Moreover, the accumulation of the product within the cell effectively limits the yield obtainable from a given mass of cells. Gilbert et al. (U.S. Pat. No. 4,411,994) report obtaining the excretion of insulin through the cell membrane of an E. coli host by fusing the insulin gene to a portion of the E. coli penicillinase gene containing the signal peptide coding sequence. However, the reference does not indicate that the polypeptide was secreted from the cell into the medium.
A considerable amount of effort has been expended by researchers to find expression systems (i.e., vector/host combinations) which will overcome the aforementioned drawbacks associated with the use of E. coli as a host for the production of heterologous (foreign) polypeptides and proteins. In particular, the art has been searching for expression systems which use non-pathogenic host organisms and which provide for expression and secretion of the desired product into the culture medium, thus simplifying recovery and increasing yields. Most of these efforts have been directed toward the construction of expression vectors containing DNA sequences which encode prokaryotic signal peptide sequences fused to the amino acid sequences of the desired polypeptides or proteins. The signal peptide is usually a sequence of about 15 to 30 residues in length, having a positively charged amino-terminal domain followed by a hydrophobic domain, which transports the attached polypeptide or protein to the cell membrane where it is deposited into the secretory pathway. By expressing the desired polypeptide or protein as a fusion with a signal peptide which is normally associated with a protein that is secreted by a prokaryotic organism, it is hoped that the prokaryotic host organism will similarly process the heterologous protein which is fused to the signal peptide. This technique has only met with limited success. For example, Palva and coworkers have reported obtaining the secretion of .alpha.-interferon from B. subtilis by fusing the .alpha.-interferon gene to a fragment of the B. amyloliquefaciens .alpha.-amylase gene containing the signal peptide coding sequence (Gene, 22:229-235 [1983]). The product was recoverable from the medium only in low yield.
To date, research directed toward the development of effective secretion systems has concentrated on the use of host microorganisms of the genus Bacillus, particularly Bacilli of the species subtilis, which are non-pathogenic and have a well-developed secretion mechanism. Bacillus species produce large quantities of extracellular protease during postexponential growth. The most abundant of these enzymes are an alkaline serine protease (subtilisin) and a neutral metalloprotease. The physiological role of subtilisin has been debated for some time.
The product of the subtilisin gene presents an important potential obstacle to the development of systems, based on Bacillus species, for the synthesis and secretion of foreign proteins encoded by recombinant genes. The inability to recover secreted foreign proteins from Bacilli in acceptable yields has led to speculation that the proteins may be synthesized at high levels only to be degraded quickly by bacterial proteases.